Name: the neuroblast assay: an assay for the generation and enrichment of neuronal progenitor cells from differentiating neural stem cell progeny using flow cytometry
Uploaded: 2012-04-22T14:00:00
Description: Watch this Scientific Journal Video about The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytomet

This work was supported by funding from the Overstreet Foundation.

1. Basic Set Up Before Proceeding to Cell Culture
<ol>
 <li>NeuroCult NSC Basal Medium and NeuroCult NSC Proliferation Supplements are mixed at a 9:1 ratio, respectively, to make complete NSC medium.</li>
 <li>A 37 &deg;C water bath is used to warm up the medium.</li>
 <li>Appropriate amount of heat inactivated fetal calf serum (FCS) is thawed.</li>
 <li>Appropriate amount of stock growth factor solutions including epidermal growth factor (EGF at 10 &mu;g/ml), basic fibroblastic growth factor (b-FGF at 10 &mu;g/ml) and...

<ol>
	<li>Azari, H., Rahman, M., Sharififar, S., Reynolds, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Expansion+of+the+Adult+Mouse+Neural+Stem+Cells+Using+the+Neurosphere+Assay.">Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay.</a> J. Vis. Exp. (45), e2393 (2010).</li><li>Azari, H., Sharififar, S., Rahman, M., Ansari, S., Reynolds, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+Embryonic+Mouse+Neural+Stem+Cell+Culture+Using+the+Neurosphere+Assay.">Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay.</a> J. Vis. Exp. (47), e2457 (2011).</li><li>Siebzehnrubl, F. A., Vedam-Mai, V., Azari, H., Reynolds, B. A., Deleyrolle, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Characterization+of+Adult+Neural+Stem+Cells.">Isolation and Characterization of Adult Neural Stem Cells.</a> Methods. Mol. Biol. 750, 61-77 (2011).</li><li>Scheffler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+and+functional+characterization+of+adult+brain+neuropoiesis.">Phenotypic and functional characterization of adult brain neuropoiesis.</a> Proceedings of the National Academy of Sciences of the United States of America. 102, 9353-9358 (2005).</li><li>Azari, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+immature+neuronal+cells+from+neural+stem+cell+progeny.">Purification of immature neuronal cells from neural stem cell progeny.</a> PLoS One. 6, e20941-e20941 (2011).</li><li>Amariglio, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor-Derived+Brain+Tumor+Following+Neural+Stem+Cell+Transplantation+in+an+Ataxia+Telangiectasia+Patient.">Donor-Derived Brain Tumor Following Neural Stem Cell Transplantation in an Ataxia Telangiectasia Patient.</a> Plos Medicine. 6, 221-231 (2009).</li></ol>

The ability to isolate defined neural cell populations (i.e. neurons, astrocytes and oligodendrocytes) might solve some of the impediments associated with the clinical application of neural stem cells (NSCs). First, it enables us to fully characterize the starting population of donor cells. Second, it allows us to use a particular cell type or a combination of different cell types with a specific ratio, depending on the nature or stage of the disease. Lastly, using highly enriched pre-differentiated neural cell progenito...

<table border="1">
 <tr>
 <td>Name of the reagent</td>
 <td>Type</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>NeuroCult NSC Basal Medium</td>
 <td>Medium</td>
 <td>Stem Cell Technologies</td>
 <td>05700</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NeuroCult NSC Proliferation Supplements</td>
 <td>Medium supplement</td>
 <td>Stem Cell Technologies</td>
 <td>05701</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>%0.05 &nbsp;&nbsp; trypsin-EDTA</td>
 <td>Reagent</td>
 <td>Gibco</td>
 <td>25300-062</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Soybean trypsin inhibitor </td>
 <td>Reagent</td>
 <td>Sigma</td>
 <td>T6522</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Pen/Strep </td>
 <td>Reagent</td>
 <td>Gibco</td>
 <td>15140-122</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>*MEM</td>
 <td>Reagent</td>
 <td>Gibco</td>
 <td>41500-018</td>
 <td>HEM component</td>
 </tr>
 <tr>
 <td>*HEPES</td>
 <td>Reagent</td>
 <td>Sigma</td>
 <td>H4034</td>
 <td>HEM component</td>
 </tr>
 <tr>
 <td>*Distilled water</td>
 <td>Reagent</td>
 <td>Gibco</td>
 <td>15230-147</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cell strainer </td>
 <td>Sieve</td>
 <td>BD Falcon</td>
 <td>352340</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>T25 flask</td>
 <td>Culture ware</td>
 <td>Nalge Nunc international</td>
 <td>136196</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>T80 flask</td>
 <td>Culture ware</td>
 <td>Nalge Nunc international</td>
 <td>178905</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>15 ml tubes</td>
 <td>Culture ware</td>
 <td>BD Falcon</td>
 <td>352096</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>50 ml tubes</td>
 <td>Culture ware</td>
 <td>BD Falcon</td>
 <td>352070</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>EGF</td>
 <td>Growth factor</td>
 <td>R&amp;D</td>
 <td>2028-EG</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>b-FGF</td>
 <td>Growth factor</td>
 <td>R&amp;D</td>
 <td>3139-FB</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Heparin</td>
 <td>Growth factor</td>
 <td>Sigma</td>
 <td>H4784</td>
 <td>Reconstituted in PBS</td>
 </tr>
 <tr>
 <td>Anti-PSA-NCAM-PE</td>
 <td>Antibody</td>
 <td>Miltenyi Biotec</td>
 <td>130-093-274</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PE- Conjugated mouse IgG1</td>
 <td>Isotype control antibody</td>
 <td>BD Pharminogen</td>
 <td>556029</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Heparin</td>
 <td>Growth factor</td>
 <td>Sigma</td>
 <td>H4784</td>
 <td>Reconstituted in PBS</td>
 </tr>
 <tr>
 <td>HrBMP-4</td>
 <td>Growth factor</td>
 <td>R&amp;D</td>
 <td>314-BP-010</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Poly-L-Ornithine</td>
 <td>Reagent</td>
 <td>Sigma</td>
 <td>P4957</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fetal Calf Serum</td>
 <td>Medium Supplement</td>
 <td>Gibco</td>
 <td>10099-141</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>GFAP</td>
 <td>Antibody</td>
 <td>DAKO </td>
 <td>Z0334</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>B-III tubulin</td>
 <td>Antibody</td>
 <td>Promega</td>
 <td>G7121</td>
 <td>&nbsp;</td>
 </tr>
</table>
*To make HEM, mix 1 x 10 L packet of MEM and 160 ml of 1M HEPES and bring the volume to 8.75 L using distilled water. Set the final PH to 7.4 and store it at 4 &deg;C.

the neuroblast assay: an assay for the generation and enrichment of neuronal progenitor cells from differentiating neural stem cell progeny using flow cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

Watch this Scientific Journal Video about The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytomet

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Research

JoVE Journal

Neuroscience

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